ABSTRACT Hexavalent chromium (Cr-6) is a firmly established human carcinogen with widespread exposure in metal- processing and other industries. There are also significant concerns about potential health effects of environmental exposure due to the presence of Cr-6 at numerous Superfund sites and its ambient and drinking water contamination. Cr-6 is a pro-carcinogen that is reduced inside the cell yielding stable Cr-3 and causing the formation of abundant Cr-DNA adducts. Cr-DNA adducts are mutagenic and genotoxic in human cells although they are weakly duplex-distorting and do not block DNA replication in vitro. We have found that genotoxicity of Cr-DNA adducts in human or mouse cells required the presence of mismatch repair (MMR) proteins. Cells lacking MMR activity were highly resistant to the induction of cell death and chromosomal damage by Cr-6. Chronic exposure to Cr-6 can therefore lead to the selection of resistant cells with inactive MMR, which provides an explanation for the very high frequency of microsatellite instability (a hallmark of inactive MMR) among lung cancers in chromate workers. Our recent findings also showed that MMR enhances Cr-6 genotoxicity via a rapid production of DNA double-strand breaks (DSB) in G2 cells immediately after replication of Cr-damaged DNA. The induction of DSB required a sequential recruitment of MSH6- and MSH3-containing complexes, indicating the unprecedented cooperation of both MMR branches in processing of Cr-DNA adducts. This application is designed to address three central questions related to the formation and the biological importance of MMR-induced DSB. These include: (1) mechanisms of DSB generation and the role of individual MMR proteins, (2) significance of error-prone and error-free pathways in repair of MMR-induced DSB, and (3) crosstalk between DSB processing and checkpoint signaling. The completion of the proposed work should identify the key processes involved in the formation and repair of highly toxic DNA lesions responsible for chromosomal breakage and other adverse genetic effects resulting from human exposure to Cr-6. Elucidation of molecular mechanisms involved in the unique processing of Cr-damaged DNA is also expected to uncover novel functions of MMR, a key genome maintenance system and a major factor in the clinical efficacy of platinum-based and some other DNA alkylating chemotherapeutic drugs.